Allergens Are Not Detected in the Bronchoalveolar Lavage Fluid Of

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4. Spierings NM, Natkunarajah J, Bansal A, Ostlere L. Should we be prescribing isotretinoin to patients with peanut allergies? Allergens Are Not Detected in the Bronchoalveolar Clin Exp Dermatol. 2015;40:824-5. Lavage Fluid of Patients Undergoing Fiberoptic 5. Kukkonen AK, Pelkonen AS, Makinen-Kiljunen S, Voutilainen Bronchoscopy H, Makela MJ. Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled Rueda M1, López-Matas MA2, Agustí C3, Lucena C3, Carnés J2, study. Allergy. 2015;70:1239-45. Valero A3,4 6. Holzhauser T, Wackermann O, Ballmer-Weber BK, et al. 1Allergology Service, Hospital Quirónsalud, Barcelona, Spain Soybean (Glycine max) allergy in Europe: Gly m 5 (beta- 2R&D Department, Laboratorios LETI S.L.U., Tres Cantos, conglycinin) and Gly m 6 (glycinin) are potential diagnostic Madrid, Spain markers for severe allergic reactions to soy. J Allergy Clin 3Neumology and Allergy Service, Hospital Clinic, Barcelona, Immunol. 2009;123:452-8. Spain 7. Sicherer SH, Sampson HA, Burks AW. Peanut and soy allergy: 4Institut d’Investigacions Biomèdiques August Pi i Sunyer a clinical and therapeutic dilemma. Allergy. 2000;55:515-21. (IDIBAPS), Centro de Investigaciones Biomédicas en Red de 8. Awazuhara H, Kawai H, Baba M, Matsui T, Komiyama A. Enfermedades Respiratorias (CIBERES), Barcelona, Spain Antigenicity of the proteins in soy lecithin and soy oil in soybean allergy. Clin Exp Allergy. 1998;28:1559-64. J Investig Allergol Clin Immunol 2019; Vol. 29(2): 148-150 9. Alden K, Chowdhury MMU, Williams PE, Kalavala M. Protocol doi: 10.18176/jiaci.0353 for investigation of possible soya allergy in patients being considered for treatment with isotretinoin or alitretinoin. Key words: Bronchoalveolar lavage. Allergen. Mass spectrometry. Clinical and Experimental Dermatology. 2016;41:326-7. Monoclonal Antibodies. Small airway. 10. Spierings NMK, Bansal A, Ostlere L. Peanut allergy and isotretinoin: reply to McCarthy et al. Clin Exp Dermatol. Palabras clave: Lavado broncoalveolar. Alérgeno. Espectrometría de masas. Anticuerpos monoclonales. Vías aéreas inferiores. 2017;42:805.

Allergen sources range in size from 0.28 to 0.40 mm for mites, 2 to 500 µm for molds, and 2 to 200 μm for Manuscript received August 17, 2018; accepted for publication November 13, 2018. pollens. Early studies suggested that given their large size, aeroallergenic sources were unable to reach the lower Christine Breynaert airways [1]. The nose filtering system retains particles Department of General Internal Medicine larger than 10 μm, and only fine or ultrafine particles reach Allergy and Clinical Immunology distal bronchioles (diameter 1-5 µm) [2,3]. However, it has University Hospitals Leuven, Belgium been demonstrated that grass pollen allergens are released E-mail: [email protected] as breathable aerosols, the atmosphere contains allergen- carrying, plant-derived, paucimicronic particles (2-5 µm) [4], and small spores and fragments of mold spores can reach the lower airways [5]. Ferguson et al [6] identified the major allergen from Dermatophagoides pteronyssinus (Der p 1) in the small airways of allergic patients. The authors assessed the link between environmental exposure to mites and the presence of the allergen in bronchoalveolar lavage (BAL) fluid in 9 patients sensitized to D pteronyssinus. Horvath et al [7] studied deposition of inhaled pollens in the airway using computer simulation. Their results suggested that pollen particles (0.5-20 μm) may deposit more efficiently in the asthmatic lung than in the healthy lung, especially in the bronchial region. The aim of our study was to examine the possible presence of major allergens from mites, pollen, and molds in BAL fluid collected from patients undergoing fiberoptic bronchoscopy. We performed an observational descriptive study. BAL samples were obtained from patients undergoing fiberoptic bronchoscopy (hospitalized and outpatients) over 1 year. Indications for fiberoptic bronchoscopy included hemoptysis, chronic cough, and upper airway assessment. Patients with

asthma were also included. The study protocol was approved Our study was designed to address the presence of allergens by the Ethics Committee of Hospital Clínic, Barcelona, Spain. in the small airways by examining BAL samples obtained from Fiberoptic bronchoscopy with BAL was performed following patients undergoing bronchoscopy to assess various respiratory the recommendations of the Spanish Respiratory Society. conditions. BAL is a bronchoscopic technique commonly used Briefly, lavage consisted of infusing 3 aliquots (50 mL each) of to diagnose lung diseases. For research purposes, this technique sterile saline solution into 1 of the subsegments of the middle is also used to sample epithelial lining fluid components and lobe or lingula. The first aliquot was collected for this study to determine the protein composition of airway content. The and stored at –80ºC until analysis. BAL samples were dialyzed hypothesis was that if major allergens were detected in BAL, and lyophilized to avoid loss of sensitivity due to dilution. For then they would indicate the capacity of these particles to reach protein profiling, 100 µg of samples lyophilized before and the small airways. However, none of the tested allergens were after dialysis were run on SDS-PAGE. The major allergens detected in the BAL samples. Phl p 5, Alt a 1, and Der p 1 were quantified using an ELISA The presence of Der p 1 [4] has been described in BAL from sandwich assay (Indoor Biotechnologies). Two samples were asthma patients, although it is not known whether this allergen analyzed using mass spectrometry (Orbi-LTQ Velos-Pro) and occurs in the general population. As far as we know, this is the compared with the peptide databases Homo, Viridiplantae, first study to address the presence of major allergens in the Fungi, and Arthropoda using the software Proteome Discoverer small airways of the general population. Although, our study 1.3 (Proteomic Service, CBMSO, Madrid, Spain). should ideally have included healthy individuals, the number BAL samples were collected from 48 patients. The reasons of patients examined and their heterogeneous nature suggest for bronchoscopy were assessment of noninfectious pulmonary that this sample could well reflect the general population. The conditions, mainly bronchial neoplasias (13 patients), patients lived in a non–allergen-free environment. In our area, noninfectious lung infiltrates (17 patients), and interstitial the allergens evaluated are mainly seasonal, with maximum lung disease (6 patients). The indications for bronchoscopy in incidences in specific seasons. House dust mites peak in spring the remaining 12 patients were chronic cough, bronchiectasis, and autumn [8], fungal spores in summer and autumn [9], and acute respiratory distress syndrome, asthma, chronic grass pollen in spring and summer. To ensure the presence of obstructive pulmonary disease, allergic bronchopulmonary allergens in the environment, we collected BAL samples over aspergillosis, and amyloidosis. a whole year. Protein profiles were similar for all tested samples. The Analyzing BAL is technically challenging, thus partially main differences observed between samples were in their explaining why we were unable to identify airborne antigens. protein quantity. All samples showed 2 main bands at 50-60 Different particles, especially high-molecular-weight kDa, and 3 at 30, 21, and 15 kDa (Figure). None of the allergens molecules, can be lost during the liquid concentration tested (Phl p 5, Alt a 1, Der p 1) were detected using the ELISA process. Besides, not knowing the dilution of alveolar fluid kits. Mass spectrometry enabled 2684 and 6139 peptides to be in the saline instilled makes it difficult to properly determine recovered from each sample respectively. No peptides from extracellular particles. Finally, low-molecular-weight mites, plants, or fungi were detected after comparison of the compounds quickly diffuse from the alveoli to the interstitium sequences with those provided in the databases. More than and capillaries. Despite these limitations, our methodology, 99% of the peptides were components of human proteins. which included the use of monoclonal antibodies and mass The remaining peptides were homologous to unknown or spectrometry, ensured the validity of our results. Mass conserved proteins such as actin and tubulin, neither of which spectrometry mainly detected human proteins from epithelial was reported as an allergen. cells entering the lavage fluid. Other peptides corresponded to very well-conserved proteins, and differences were probably due to small variations in mass detection. Although we identified some of these peptides, none of them were kDa constituents of known allergens. 97.4 In summary, we were not able to detect major allergens 66.2 of mites, pollen, or molds in BAL. Further studies are needed 45.0 to assess potential differences in detected allergens between populations, mainly asthma patients and those with other lung 31.0 diseases. This type of information will help establish whether 21.5 or not it is necessary for an allergen to reach the distal airways to trigger an asthma attack. 14.4 Funding St 35 29 39D 23 23D 4 31D 31 35D This study was funded by Societat Catalana d'Al·lèrgia. Figure. SDS-PAGE with a selection of samples. One hundred micrograms of lyophilized powder was run in each lane. The numbers under each lane Conflicts of Interest correspond to the number of the patient sample. Lines with numbers only MA López-Matas and J Carnés are employees of correspond to predialysis samples, and lines with numbers followed by a D correspond to postdialysis samples. St indicates molecular weight Laboratorios LETI. The remaining authors declare that they standard low-range (Bio-Rad). have no conflicts of interest.

References Actin Allergen of Common Periwinkle Sea Snail 1. Shah R, Grammer LC. Chapter 1: an overview of allergens. (Littorina littorea) Allergy Asthma Proc. 2012;33 Suppl 1:S2-5. 2. Wilson AF, Novey HS, Berke RA, Surprenant EL. Deposition of González-Mancebo E1,5, Trujillo-Trujillo MJ1,5, Gandolfo- inhaled pollen and pollen extract in human airways. N Engl J Cano M1,5, Mohedano-Vicente E1,5, Cuesta-Herranz J2,5, Med. 1973;288:1056-8. Bartolome B3*, Pastor-Vargas C4,5* 3. Cheng Y, Smith SM, Yeh H. Deposition of ultrafine particles 1Allergy Unit, Hospital Universitario de Fuenlabrada, IdiPAZ, in human tracheobronchial airways. Ann Occup Hyg. Madrid, Spain 1997;41:714-8. 2Allergy Department, IIS-Fundación Jiménez Díaz, UAM, Madrid, 4. Taylor PE, Flagan RC, Valenta R, Glovsky MM. Release of Spain allergens as respirable aerosols: A link between grass pollen 3R+D Department, Roxall, Bilbao, Spain and asthma. J Allergy Clin Immunol. 2002;109:51-6. 4Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, 5. Rick EM, Woolnough K, Pashley CH, Wardlaw AJ. Allergic Madrid, Spain Fungal Airway Disease. J Investig Allergol Clin Immunol. 5ARADyAL Network, Instituto de Salud Carlos III, Madrid, Spain 2016;26(6):344-54. *Both authors should be considered last authors 6. Ferguson P, Broide DH. Environmental and bronchoalveolar lavage Dermatophagoides pteronyssinus antigen levels in J Investig Allergol Clin Immunol 2019; Vol. 29(2): 150-152 atopic asthmatics. Am J Respir Crit Care Med. 1995;151:71-4. doi: 10.18176/jiaci.0356 7. Horvath A, Balashazy I, Farkas A, Sarkany Z, Hofmann W, Czitrovszky A, et al. Quantification of airway deposition Key words: Shellfish allergy. Mollusk allergy. Periwinkle allergy. Actin. of intact and fragmented pollens. Int J Environ Heat R. Tropomyosin. 2011;21:427-40. Palabras clave: Alergia a marisco. Alergia a moluscos. Alergia a bígaro. 8. Demoly P, Matucci A, Rossi O, Vidal C. A year-long, fortnightly, Actina. Tropomiosina. observational survey in three European countries of patients with respiratory allergies induced by house dust mites: Methodology, demographics and clinical characteristics. BMC Pulm Med. 2016;16:85. 9. Gomez de Ana S, Torres-Rodríguez JM, Ramírez EA, García Shellfish are one of the main food allergens in adults SM, Belmonte-Soler J.J. Seasonal distribution of Alternaria, and include a diverse group of species with shells or shell- Aspergillus, Cladosporium and Penicillium species isolated like exo- or endoskeletons. They comprise both crustaceans in homes of fungal allergic patients. J Investig Allergol Clin and mollusks. Mollusks include gastropods (limpets, snails, Immunol. 2006;16:357-63. winkles, and abalone), bivalves (mussels, clams, razor fish, oysters, and scallops), and cephalopods (squid, cuttlefish, and octopus). Shellfish are an important food source and are consumed throughout the world [1]. Japan and other coastal regions of Asia are major mollusk consumers [1]. Countries in southern Europe (Spain, France, Italy, and Portugal) also Manuscript received October 17, 2018; accepted for publication consume high quantities of mollusks and crustaceans [1]. November 14, 2018. Mollusk allergies have been documented with gastropods, bivalves, and cephalopods [1]. Although the importance of Jerónimo Carnés mollusk allergy is becoming increasingly recognized, its Research & Development Laboratorios LETI, S.L.U. prevalence is unknown. Mollusks were responsible for 5.6% E-mail: [email protected] to 11.7% of severe allergic reactions in France during 2002 and 2003 and are reported to have caused 36% of cases of anaphylaxis in Singapore during the same period [1]. The common periwinkle (Littorina littorea) is a marine gastropod mollusk that has gills, an operculum, and a dark and sometimes banded shell. It is a member of the Littorinidae family. Common periwinkles are native to the northeastern coasts of the Atlantic Ocean (northern Spain, France, England, Wales, Scotland, Ireland, Scandinavia, and Russia). They are eaten in coastal areas of Spain, Scotland, England, Wales, and Ireland. Periwinkles are highly valued in African and Asian cuisine. The meat is high in protein and ω-3 fatty acids and low in fat (http: wikipedia.org/wiki/Common_periwinkle). We report the case of a 49-year-old woman who developed symptoms after consuming periwinkles. Two hours after eating cooked periwinkles (approximately 250 mg) and